TW200529866A - Cysteine rich peptides for improving thiol homeostasis - Google Patents

Abstract

Described is the use of a mixture of peptides, the peptides comprising at least 6.5 %wt cysteine, for the manufacture of a medicament, supplement, beverage or food product for restoring thiol homeostasis. Furthermore, a method is described for restoring thiol homeostasis in a subject in need thereof, said method comprising administering to said subject an effective amount of a mixture of peptides, the peptides comprising at least 6.5 %wt cysteine.

Description

200529866 IX. Description of the invention: [Technical field to which the invention belongs] This invention refers to the use of the skin mixture in the manufacture of drugs, supplements, drinks or food products that restore the body balance of thiols. These peptides contain at least 6.5 % By weight of cysteine. [Prior art] * The physiological activities of a variety of essential proteins, including enzymes, depend on the oxidation-reduction state of oxidation-reduction-sensitive thiol groups, that is, these thiol groups are reducing GSH) or oxidized (_s_s_ ) State exists. These proteins react when there is little change in the redox potential of their environment. The entire network of non-protein biological thiol antioxidants, antioxidant vitamins, and antioxidant enzymes is important in maintaining the structure of the protein or in establishing the redox signal transfer to the corresponding target. The body's so-called thiol buffers include GSH, GSH precursors, and cysteine, as well as all redox-sensitive thiol groups in proteins. The treatment of these thiols with several antioxidant enzymes is very complex and is responsible for the reduction of the structure and function of proteins, such as enzymes, and the balance of oxidation reactions. Redox chain effects, for example, the delivery of certain #transductioon chains such as the NF_kB / p50 system, receptor functions, protein kinases and phosphatases, and the delivery of serum albumin (such as those carrying fatty acids) The function is based on the longevity of the thiol bound to thiol, and possibly a protein of apoptic machin ^ y. In modern day life, almost everyone is exposed to the chemical & Atmospheric pollution, exposure to automobile exhaust, smoking, drug abuse, alcohol consumption, and coffee and / or drug use can cause excess amounts of chemical compounds in the body 99317.doc 200529866. Intracellular thiols are molecularly used in the most important redox manner to effectively remove adverse effects on the body caused by excess in vitro chemicals. The aforementioned mechanisms involved in the redox state and balance regulated by thiols are included in the '' thiol homeostasis, 'within the term. The term is known in the art (see, for example, D.M. Townsend, K.D. Tew and H. Tapiero, Biomed.

Pharmacother, 2004, vol. 58 (1): 47-55). [Summary of the Invention] It has now been surprisingly discovered that the peptide mixture has a peptide content of at least 6.5% by weight based on the protein portion of the peptide product. It works, whether he is suffering from one or more diseases or seems to be very healthy. As used herein, the% weight ratio is related to the weight of cysteine in the total peptide weight in the peptide mixture. The latter can be calculated by known methods used in the art, that is, 6.38 times the total weight percentage of nitrogen in the peptide mixture. Therefore, the present invention relates to the use of peptide mixtures in the manufacture of pharmaceuticals, supplements, beverages, or food products that restore the homeostasis of thiols, and such peptides contain cysteine at least 6.5% by weight. It has been found that for people, especially those over 50 years of age, such thiol homeostasis caused by the peptide mixture can improve vitality and viability, relieve fatigue and stress, improve sleep quality and enhance Mental alertness. As described herein, a peptide is defined as: a derivatized acid chain derived from one or more proteins; the molecular weight of the peptide is preferably between 2000 & It is between 300 Da and 6,000 Da, particularly preferably between 400 Da and 5,009999317.doc 200529866

Da. Preferably, the peptides in the peptide mixture contain at least 6.5% by weight, more preferably at least 6.7% by weight, particularly preferably at least 6.9% by weight, and most preferably at least 7% by weight. Cysteine. On the laboratory scale, it has been found that the content of cysteine of peptides can be as high as 20% by weight; however, the cysteine content of peptide mixtures produced in large quantities by current technology is 6. 5-7. This is also the better range now. However, it is preferred that the cysteine content of the peptide should be as high as possible to provide more flexibility when used and administered. When used in medicine or supplements, the preparation can be combined with any suitable carrier, diluent, adjuvant, excipient, etc. to make the drug in the desired administration form. The drug or supplement is advantageously administered orally. The term "supplement" includes food supplements and health products such as health drinks. I. When used for purpose, the skin-winning mixture, which contains at least 65% by weight of cysteine, can be administered alone or in combination with a pharmaceutically acceptable carrier. This can be known methods, such as, Remington, pharmaceutical

Sciences Handbook ", Mack Pub · Co ·, Ν ······, the method described in the example of 'made preparations are tablets, capsules, syrups, etc. for oral administration' and forms for parenteral administration Is an acceptable liquid sterilization solution or suspension, implant, etc. When used in beverages or food products, the peptide mixture contains at least 6 · 5 ° /. The weight ratio of cysteine can be combined with any general food ingredient. The term "beverage" includes irritating beverages and syrups, and dry powders that are soluble in other beverages for the preparation of ready-to-drink beverages. 99317.doc 200529866 In a preferred embodiment, the peptide mixture, the peptide Contains at least 6.5% cysteine for the preparation of supplements, beverages or food products. These supplements, beverages or food products are 1 mg 000 mg of cysteine per day, compared to It is preferably 50-60 mg, more preferably 80-30 milligrams, and most preferably 100-200 mg. After administration, it was found that the peptide was mixed to make the seemingly healthy or suffer from specific Patients with health problems feel energetic and make the overall situation better. In the preferred embodiment, at least 70%, preferably at least 80% of the peptides _ 纟 at least the terminal cysteine, this cysteine Amino acids are often found in the human or animal body to restore the homeostasis of thiols. That is, at least 70%, preferably at least 80% of the peptides have one or two terminal cysteine. Terminal The cysteine content can be known by this technique, for example Preferably, the peptide mixture contains at least 6.5% by weight of cysteine, at least 60%, preferably at least 70%, and more preferably at least 80%. % Of cysteine in the form of cystine. The so-called " cystine, here is the name of the cysteine in the oxidized form, that is, a semi-photoamine residue with a sulfur bridge and Another cysteine residue is coupled. Throughout this description, "cysteine ,, • The term refers to reduced (with free SH-groups) and oxidized (cysteine) • Cysteine acid. Free-state thiols like cysteine are susceptible to oxidation in the body resulting in free radicals. That is, high doses of thiol have the effect of pro-oxidants after entering the bloodstream. Therefore, it is preferred that the cysteine residues in the mixture exist in an oxidized form, as this form has less chemical reactions and is therefore safer for individuals than free cysteine (see, for example, BiotWols in Health and Dlsease, L 加加 —E 义 池 ⑽), 99317.doc 200529866

Marcel Dekker Inc "New York, Basel, Hongkong (1995)). In another specific embodiment, the peptide mixture, which contains at least 65% by weight cysteine peptide, is used for prevention and / Or reduce the effect of drinking. An important intermediate in the process of alcohol conversion is acetaldehyde, which is a highly toxic compound and has high chemical reactivity to proteins, DNA and lipids that make up the body. The production of acetaldehyde is The body has long-term and short-term negative effects. It is generally believed that thiol groups can react directly with acetaldehyde without enzymatic intervention in order to ensure the elimination of this compound and thereby reduce its serious consequences for the body. It is now believed that giving The peptide mixture rich in cysteine will lead to the restoration of redox and the internal balance of thiol, so that there are sufficient thiol groups in the body to remove acetaldehyde from t. This will not only prevent or reduce alcohol consumption The short-term effects of the body, such as hangovers or flushing, also reduce the long-term effects, such as liver dysfunction. Therefore, in another specific embodiment, it contains at least 65% by weight of cysteine. For this peptide Prevent and / or reduce hangovers. It is now believed that after ingesting the peptide mixture of Hikata Waseda Cysteine acid, the thiol balance is restored in the body, so there are sufficient thiol groups to react with acetaldehyde. A compound thereby reducing its effects on the body, especially hangover. In yet another embodiment, the peptide mixture, the peptide contains at least 6.5% of the replacement ratio of cysteine for prevention and prevention / Or reduce facial flushing. Many people, especially Asians, have genetic characteristics in the activity of acetaldehyde dehydrogenase (ALDH) required for the oxidation of acetaldehyde, a toxic metabolite of alcohol. Therefore, they lack ALDH There is acetaldehyde accumulation in the body, which causes facial flushing and other cardiovascular symptoms (Η.Μ. Chao, Alcohol Clin. Exp · Res · 1995. vol · 19 (1) ·· 99317.doc -10- 200529866 104-109 ). This type of person can experience facial flushing even after ingesting a small amount of alcohol, which is embarrassing. It has been found that ingestion of the peptide mixture can prevent and / or reduce facial flushing and other effects caused by lack of ALDH, individuals Feel more confident and less awkward in a public environment. In a specific embodiment, the peptide mixture, which contains at least 6.5% by weight of cysteine, is used in the manufacture of pharmaceuticals, supplements, beverages or food products for enhanced vitality. It has now surprisingly been found that even Individuals who have not been diagnosed with any health problems also feel energetic after ingesting the peptide mixture. The individual generally feels better, more energetic, and more adaptable to life. It is believed that this increased vitality is due to thiols and redox in the body Reasons for improved balance. In yet another embodiment, the peptide mixture, the peptide contains at least 6.5% by weight of cysteine, for the manufacture of medicines, supplements, beverages or food products for prevention and / or Reduce fatigue. As noted above, individuals generally feel more energetic and less tired. In an attractive embodiment, the peptide mixture of the present invention is also used to reduce the symptoms of chronic fatigue. In yet another specific embodiment, the peptide mixture contains at least 6.5% replacement ratio of cysteine and is used in the manufacture of drugs, supplements, beverages or food products for improving sleep. Individuals who ingested the peptide mixture experienced better sleep ', which appears to be due to the more effective removal of unbalanced chemicals by restoring thiol's homeostasis. In yet another embodiment, the peptide mixture contains at least 6.5% cysteine by weight, and is used in the manufacture of drugs, supplements, beverages or food products to prevent the occurrence of metabolic symptoms. , Especially to prevent the occurrence of non-insulin-dependent diabetes mellitus (NIDDM). Metabolic symptoms are believed to be caused by genetic structure 99317.doc -11-200529866 and lifestyle habits such as diet and activity levels. In general, symptoms caused by metabolic problems develop over time. When an individual is obese and has high blood pressure, there is already a risk of further development of metabolic symptoms. Metabolic symptoms begin when insulin loses its ability to allow cells in the body to absorb glucose from the blood, resulting in long-term maintenance of high glucose levels. Individuals are at risk of developing non-insulin-dependent diabetes due to prolonged high glucose levels. Various stages of NIDDM, namely insulin resistance, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, and severe loss of stone-cell function (obvious NIDDM), are associated with multiple toxicities. It is believed that administration of the peptide mixture of the present invention will restore thiol homeostasis, thereby preventing and / or reducing insulin resistance poisoning due to metabolic symptoms, especially poisoning caused by thiol-induced protein cross-linking or DNA modification (MR · Hyden and sc Tyagi

Pancreas 2002, vol. 3 (4): 86-108). In another specific embodiment, the skin-mixing mixture of the present invention is used for preventing and / or reducing the development of cardiovascular disease in metabolic symptoms, especially atherosclerotic arteriosclerosis and the intermediate state of NIDDM. This is due to the various toxicity of the aforementioned niddm (M.R. Hayden and S.C. Tyagi, Atheroscleropathy Cardiovasc. Diatetol. 2002, vol. 1 (1): 3). In another embodiment, the 'skin mixture of the present invention is used to lower blood pressure. ACE converts angiotensin I into angiotensin π. The latter is an effective vasoconstrictor ' that leads to an increase in blood pressure when poorly regulated. Nowadays, because thiol balances the inhibition of ACE activity in the body, angiotensin production is reduced, thereby ensuring lower blood pressure or preventing an increase in blood pressure. Therefore, the use of the peptide mixture of the present invention can also help to further prevent the occurrence of metabolic symptoms ((R. 99317.doc -12- 200529866

Bataller, RF Schwabe, ΥΥΗ Choi, L. Yang, ΥΗ Paik, J. Lindquist, T. Qian, R. Schoonhoven, CH Hagedorn, J J. Lemasters, and DA Brenner. J. Clin. Invest. 2003 , vol. 112 (9): 1383-1394) 〇 In another specific embodiment, the peptide mixture, the peptide contains at least 6.5% by weight of cysteine, for the manufacture of drugs, supplements, beverages or Food products are intended to prevent and / or treat drug-induced poisoning. It is believed that the correct redox state of thiol and redox homeostasis is restored to ensure that drugs affecting the liver are eliminated more quickly to avoid and / or treat drug-induced poisoning. The peptide mixture, which contains at least 6.5% by weight of cysteine, is used in the manufacture of pharmaceuticals, supplements, beverages or food products for whitening the skin. The main factor determining skin color is the concentration and mixing ratio of various melanin in individual pigment cells, namely, true melanin (black / brown pigment) and pheomelanin (amber / red pigment). It is now believed that the peptide mixture regulates tyrosinase activity, one of the early enzymes that converts melanin, reducing the production of melanin and causing the skin to turn white. In addition, it is now believed that the thiol group in the cell can adjust the ratio of true melanin to chromaffin melanin, which is beneficial to chromaffin melanin, resulting in lighter pigmentation. Because the peptide mixture also increases the intracellular thiol concentration, the synthesis of melanin can be changed to make a lighter chromaffin melanin. The ratio of these two pigments in the skin is 1; 1 is particularly expected to achieve visual effects. The peptide mixture of the present invention can be used for oral treatment of local skin fading, such as scarring of darker skin after acne inflammation (RM Haider, HL Brooks, and VD Callender, Dermatol. Clin. 2003, vol. 21 ( 4): 609-615). 99317.doc 13 200529866 In another embodiment, the peptide mixture, the peptide contains at least 6.5% by weight of cysteine, for reducing inflammation. The inflammation process itself is accompanied by the need for sulfur-containing amino acids to maintain acute phase protein synthesis and immune cell activity to counteract the generation of free radicals caused by inflammation and later restore the damaged tissues of the fork. Chronic inflammation can lead to dying of the thiol and oxidant internal balance, with serious systemic consequences. It is believed that the peptide mixture of the present invention produces beneficial effects in inflammatory diseases such as arthritis, chronic inflammatory bowel symptoms, acne, and sepsis (F. Santangelo, Cmr. Med · Chem · 203, vol. 10 (23): 2599-2610). In a preferred embodiment, the peptide mixture, which contains at least 65% by weight of cysteine, is prepared by the following steps: a) cleaving a protein source protein into a peptide; b) using The peptide obtained in step a) of at least one exo-peptidase digestion has the effect of attenuating at least the cysteine position in the peptide to generate a peptide having terminal cysteine; c) purification and digestion Peptide. The protein of protein origin is cleaved into smaller peptides in step a). Such cleavage can be accomplished by cleavage reactions known in the art; it is preferred to use, for example, endo-peptidase to hydrolyze the peptide bonds of the protein to produce cleavage to produce a peptide of the required length, and to increase the exo-peptidase The amount of substrate required. An example of an endopeptidase suitable for the purposes of the present invention is Alcalase from NOVO Nordisk. In the second step, the peptide obtained by the cleavage reaction is digested with at least one exo-peptidase. Here, the meaning of "at least one exo-peptidase" is that the digestion reaction can be completed with one or more different exo-peptidases. Exo-peptidase releases a single amino acid from the terminal of the peptide 99317.doc -14- 200529866 In addition, the peptidase and digestion reaction conditions are selected such that the effect of the peptidase on at least the cysteine position of the peptide is attenuated. The term "minimum attenuation" means that the peptidase is not under the selected reaction conditions Remove cysteine from the peptide or have a lower preference for cysteine cleavage. Therefore, compared with the amino acid cleaved from the peptide, the cleavage reaction is slower. With the use of this exo-peptidase and reaction conditions, a peptide can be produced, the terminal amino acid of which has been removed, and only the cysteine residues are left. An enzyme with an exopeptidase function is found only on cysteine. It should be understood that such peptides may have one or more amino acid chains on the amine & L chain with each other as cysteine. The disulfide bridges of the amino acids are coupled. So "digested with terminal cysteine Anti-peptide ,, reflected the fact that at least one terminal such multi-chain peptide has a terminal cysteine & C & L. Naturally, such peptides may contain more than one terminal cysteine. Preferably, the enzyme activity is rendered inactive before the purification step by, for example, a pH change or heat inactivation treatment. Preferably, the exo-peptidase contains carboxy-peptidase Y (EC 3.4161), and it has been found that this enzyme can effectively attenuate the terminal cysteine residue, thereby producing a peptide having a terminal cysteine residue . The cleavage step a) and the digestion step b) can be performed simultaneously. For example, endo-peptidase and exo-peptidase can be used under the same reaction conditions. In addition, an enzyme preparation having two activities of endo-peptidase and exo-peptidase can also be used. Finally, this digested peptide is purified. A suitable method for separating and digesting peptides by free-form amino acids released by exo-peptidase is known in the art. Due to the difference in the molecular weight of amino acids between the peptides containing cysteine and the free state released by the action of exo-peptidase 99317.doc -15- 200529866, this difference can be used to purify cysteine-rich peptides. Peptide. Several techniques known in the art can be used for this purpose. Preferably, the free-form amino acid and other low molecular weight compounds are separated by a membrane step, preferably by ultrafiltration, diafiltration, or microfiltration, or nanofiltration. The purification process may also include an immobilized metal affinity chromatography analysis step (IMAC), according to Kronina et al., Journal of Chromatography A, 852 (1999) pages 261-272. The cysteine-rich peptide was then dried as follows. # In a specific embodiment, the outer peptidase and cleavage reaction of step b) are selected in such a way that the outer peptidase is attenuated less than the cysteine of the peptide. This can be done on digested peptides with apparent terminal cysteine digestion. The protein source may be any source as long as it includes a cysteine-containing protein. Protein sources can also be prepared before using the method of the present invention, such as preparing two or more protein sources before or during lysis. Preferably, the protein source is composed of edible protein so that the digested protein can be used as a food supplement. In a very specific embodiment, the protein source includes whey protein isolate (WPI) and / or whey protein concentrate (WPC). The terms "whey protein isolate" and "whey protein concentrate" are known in the art. The whey protein concentrate is a protein product containing 35-80% by weight, and the protein content of the whey protein isolate is 90% w / w or more. An example of WPC 80 is Alacen 132 of Tatua (New Zealand); an example of WPI is Bipro of Davisco Foods International (USA), or ARLA Foods, Denmark's Acid Whey Protein Isolate. The whey protein isolate contains very suitable cysteine-rich proteins, such as albumin, especially α-99317.doc -16- 200529866 ritual albumin and bovine serum albumin. These proteins can be used as starting protein sources. In another preferred embodiment of the method of the present invention, the protein source includes — protein, specifically | county π a 丨, main ... $ 夕 种 由 白 寻 J 疋 α _ Li> moon protein, bovine serum Protein, ^, ϊμ π. _. ^ Protein (eg egg + cystamine @ 文 proteinase inhibitor), wheat gluten,

At Zein Isolate, r-Lupin (Lupin), and Bunch. Ding Yue protein

Preferably, steps a) and b) are carried out in a form that allows sulfur bridges between the semi-photogenic amino acid residues in the protein of the protein source to be oxidized as much as possible. In this way, a mixture of peptides rich in cysteine is obtained. Most of the cysteine residues are oxidized and coupled to other peptides via a disulfide bridge. The oxidized peptide mixture is less reactive and therefore more stable in use. Another advantage is that most enzymes with exo-peptidase activity do not cleave the oxidized cysteine, and the reduced form of cysteine is cleaved from the peptide by the enzyme. Low activity. To avoid modification of the chain in the peptide mixture, steps a) and b) are preferably performed between 1) 112 and 8. The hydrolysis process is preferably performed in an acidic environment. The disulfide bridge is more stable in acidic yang than in alkaline environment. [CreightOne, TE ·, 1993, fine linen structures and Molecular Properties. 2nd Ed .; Freeman and Company, New York] In a very attractive embodiment, the enzyme with endopeptidase function also has Exepeptidase function, its exepeptidase function is attenuated at the cysteine position. Such enzymes are known in the art and have the advantage that steps a) and b) can be performed simultaneously. Examples of enzymes with two functions of endo-peptidase and exo-peptidase are 99317.doc 17 200529866

Flavourzyme (NOVO Nordisk), Acid Protease A, Protease M, Protease 2A? Protease B (Amano Enzyme), Corolase PN-L (AB Enzymes, UK), Enzeco Acid Fungal Protease (EDC, USA) or a combination of two or more of these Thing. Preferably, at least 70% of the peptides in the preparation, more preferably at least 80% of the peptides contain terminal semi-photoamines, which are convenient for human or animal body use. These terminal cysteines are prepared using exo-peptidase as described above. In yet another aspect, the present invention relates to a method for restoring thiol homeostasis in an individual in need thereof, the method comprising administering to the individual an effective amount of a peptide mixture containing at least 6.5% by weight of cysteamine acid. This method has the advantages of the above reasons. In a preferred embodiment, the method is for preventing and / or reducing the effects of drinking on individuals in need thereof, in particular preventing and / or reducing hangovers and preventing and / or reducing facial flushing. This method has the advantages of each of the above reasons. In another embodiment, the method is for enhancing vitality, in particular for preventing and / or reducing fatigue, especially chronic fatigue, for the reasons described above. This method can also be beneficially used to improve sleep. It has now been found that individuals who have used an effective amount of a peptide mixture which contains at least 6.5% by weight of cysteine, can sleep better for the same reason as described above. Alternatively, the method can be used to prevent the development of metabolic symptoms, especially non-insulin-dependent diabetes mellitus (NIDDM), and to prevent and / or reduce the development of cardiovascular diseases, especially atherosclerosis, for the reasons described above. In another embodiment, the method can be used to lower blood pressure. The reasons for the advantages of this method are as described above. 99317.doc -18- 200529866 For the prevention and / or treatment of toxicity caused by drugs, the reason is in another specific embodiment, the ancient, traditional J τ 60 Hai method can be used to whiten the skin, the reason is as above . Eighth In another specific embodiment, the ancient method and the m · τ method can be used to reduce inflammation for the reasons described above.

Preferably, in the above method, the peptide mixture containing at least 6.5% by weight of cysteine is prepared by a method having the following steps: a) cleaving a protein of a protein source into a peptide; b) the peptide obtained in step a) of the peptidase digestion, the effect of the peptidase is to attenuate at least the cysteine position of the peptide, thereby generating a digested peptide with terminal glutamic acid; c) purification Digested peptides. The resulting peptide mixture has a large proportion of terminal cysteine

This method can also be used as described above. Residues that are readily bioavailable to restore thiol homeostasis. This method can be implemented in any of the specific embodiments described above. The invention is illustrated by the following non-limiting examples. The percentages shown are by weight unless otherwise stated. [Embodiment] Examples Example 1 Method for preparing cysteine-rich peptides by adding WPI into pre-heated demineralized water and continuing heating to the temperature (50 ° C) required by this method to obtain 5% weight Specific Whey Protein Isolate (WPI;-Bipro, Davisco in general). The pH was adjusted to 3 by adding 30% sulfuric acid. Add 99317.doc -19- 200529866 ENZECO fungal protease (EDC, U.S.) to start hydrolysis. The enzyme / protein ratio is generally 2% by weight, based on dry protein. After a suitable hydrolysis time, usually 20 hours, NaOH (33%) is added until the pH becomes 6.5, and then the mixture is heated to 104 ° C and maintained at this temperature for 3 minutes. After volume reduction and microfiltration, use Nadir SS NF-PES-10 3838 B membrane module to percolate the hydrolysate with demineralized water at 50 ° C (generally 300%, sometimes 200%). This microfiltration was performed at 50 ° C. When 25% dry material is reached, the residue is spray-dried. • Example 2. Typical analysis The analysis was performed by a commercial laboratory (CCL Nutricontrol, Veghel, The Netherlands) to obtain a certificate. Method: Before the peptide was hydrolyzed by HC1, the cysteine in the sample was oxidized with performic acid, and derivatized with tristetrione as the post-column. The free amino acid was used as ion exchange chromatography. According to EC Guideline 98 / 64 / EG of 3-9-1998; Publication L257 / 14-23 dated 19-9-1998) 〇 Typical total solids 95,1 protein (N > 6,38)% 83,4 protein (N # 6, 62)% 86,0 Cysteine g / kg 65,50 Cysteine% / Prot 7,9% Alanine Alanine / kg 37,0 Arginine g / kg 18,6 99317.doc -20- 200529866 915,29 The free thiol group in the peptide is measured with DTNB (Ellmann, s reagent) in the presence of urea, and reduced with NaBH4. The SH / SS ratio is calculated based on the existing thiol concentration, and there is no reduction / total thiol concentration after reduction, and it is actually measured to be 65%. Example 3 · Molecular weight distribution method: Aspartic acid, glutamic acid, glycine, histamine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, and threonine After enzymatic hydrolysis) Tyrosine valine acid Total amino acids g / kg 146,9 g / kg 186.4 g / kg Π? 7 g / kg 19,6 g / kg 43.0 g / kg 59,4 G / kg 89,8 g / kg 12,2 g / kg 18,4 g / kg 37,6 g / kg 43,3 g / kg 47,3 g / kg 11,4 g / kg 18,6 g / kg 45,6 kg HPLC system: isocratic HPLC system with UV analyzer, autosampler 5 Waters Millenium Data Acquisition Software column: Progel TSK-G2000SWXL 7.8 mm x 30 mm (Supelco) 99317.doc -21-200529866, guard column ( guard column): Progel TSK SWXL (Supelco) ^ Eluent: 30% acetonitrile / H2O / 0, 1% TFA flow: 1 ml / min Determination: 214 nm Calibration: HPLC standard (Sigma); Carbonic anhydride, RNA Enzyme a, aprotinin, insulin, bacteriocin, phenylalanine. The measured molecular weight distribution is as follows: MW-range (D) Area ° / 〇 > 10? 000 17,5 10,000-5,000 3,7 5,000-2,000 6,6 2,000-1,000 12,1 1,000-500 20,2 & lt 500 39,9 Example 4. Injury dose of cysteine-rich peptides: 4 tablets / day, 200 mg of L-cysteine / day was administered as a peptide mixture.

Tablet weight 850 mg per 100 grams of cysteine-rich peptide 88.24 grams of microcrystalline cellulose 1 i0, 59 grams of silicon dioxide 2 0.47 grams of magnesium stearate 0.35 grams of stearic acid 0.35 G ^ vicel PH-102-FMC 99317.doc -22- 200529866 2CAB-0-SIL M-5 Premix the powder to the last minute and then add stearic acid. Money is pressed directly (pressure 20 kN, hardness 160 N). Example 5. Preparation of cysteine-rich chocolate candy

One piece of sugar is administered with 200 mg of L-cysteine in the form of a peptide mixture rich in cysteine. Per 100 g per guest (40 g) cysteine-rich peptide 8,30 g 3,32 g Maltitol Syrup1 66,01 g 26,41 g calcium caseinate 10,65 g 4,26 g chocolate liquid 8,52 g 3,41 g sodium caseinate, granules 4,26 g 1,70 g coconut oil 2,13 g 0,85 g cream, unsalted 0,11 g 0,04 g egg filling Fat 0.001 g 0.003 g vanilla flavor 0.01 g 0.03 g lycasin 85% solution Example 6. Heat-treated buttermilk beverage containing cysteine-rich peptides Preparation of 100 g of L-cysteine / guesteine peptides, 86 g, 0.05 g skim milk, 55 g bran, 6 g Maltitol, 3 g lactic acid, 0.902 g, 99317.doc -23- 200529866 Pectin 2.0, 3 g dwarf flavor 0,055 g water 34,6 g inoculum 3 0.2 g C maltidex 16385 Cerestar 2 Genu Pectine YM-115-H CP Kelco YC-X11 Christian Hansen mixes milk with water. Add cysteine-rich peptides, sugars, and continue stirring to dissolve, and then sterilize (90 ° C, 5 minutes). After cooling to fermentation temperature (42 ° C), the inoculum was added. Ferment to pH 4.3. Use lactic acid to lower the pH to 3.8-4.0. Add pectin under vigorous agitation. The mixture was heated to 70. (3, homogenize at 120/20 bar, add flavoring agent. After filling, sterilize the product (8 (TC / 3 minutes). Example 7: liver cleansing with cysteine-rich peptide (nver cieansing) Preparation of beverages 250 ml of L-cysteine / guest cysteine peptide per 100 g per guest 0,43 g 1,08 0,06 g glucose 4,4 g 11 g fruit sugar 3,6 G 9 g apple spice 0,055 g 0,14 banana spice 0,037 g 0,09 pectin 1 0,15 g 0,37 water 1 91,33 g 228,36

Genu Pectine YM-115-H CP Kelco dissolves all dry ingredients in water, adjusts 99317.doc -24- 200529866 pH to 3.8 with citric acid (+ Λ0, 14% of total), and then uses malic acid (total (+ 66% of the plant) 11 is adjusted to 3,5. Preheat the solution to 70 ° C and add the pectin premix (4% aqueous solution). After homogenization (150 bar), the product is filled and sterilized, or sterilized before filling. Example 8: Reduction of acetaminophen-induced liver poisoning after administration of cysteine-rich peptides Mice were fed an isocalodc and isonitrogenic diet containing amines at a constant concentration of sulfur Acid (38 mg / kg) or a diet supplemented with cysteine-rich peptides (62 mg / kg) for 14 days. After 14 days, animals in each diet group were challenged with acetaminophen (orally, 300 mg / kg body weight in corn oil), and then fasted for 12 hours. Immediately after challenge and after 12 and 24 hours, 6 (t = 0) and 9 rats (t = 12 hours and t = 24 hours) were taken from each group, and the rats were sacrificed, and the liver was removed for biochemical analysis and histological examination. Blood samples were taken for marker liver enzyme analysis. Both the control group and the group receiving cysteine peptides were harmful to acetaminophen, but the rat livers that continued to supply cysteine peptides were better than the control group in recovering their structural integrity. As obvious. Liver aspartate aminotransferase (ASAT) [U / L] The specific activity of the liver enzyme aspartate aminotransferase is a relevant indicator for liver injury. If the activity of this enzyme is increased, it means liver damage. Hours after challenge Casein diet plus acid peptide diet rich in cysteamine 0 69 +/- 2 78 +/- 6 12 126 +/- 14 92 +/- 15 99317.doc -25- 200529866 24 120 +/- 16 109 + / ^ 16 The above table clearly shows that hepatic damage caused by acetaminophen is obtained by ingesting a peptide rich in cysteine There is substantial relief. Necrotic cells [cells / 20 domains] Microscopic histological examination Number of hours after challenge Casein diet plus cysteamine-enriched diet Medium minimum maximum Middle minimum maximum 0 2,5 0 4 4 1 14 12 51 2 229 61 1 376 24 16 0 89 3 1

From the table above, the number of necrotic cells can be observed. After the increase at the beginning, the diet group supplemented with cysteine-rich peptides actually decreased compared with the control group (casein group). This indicates that the recovery of biothiols The reason for homeostasis is the quick recovery of tissue integrity. Vacuolar cells [cells / 20 domains] microscopic histological examination hours after excitation

Meal protein diet plus cysteine peptide-rich diet Mid-Min Max Mid-Min Max 0 16 1 146 4 0 13 12 80 16 824 419 10 618 24 11 2 141 7 0 32 vacuole cells indicate that cells are beginning to damage, the fact Vesicularization leading to cell necrosis in sigma is a reversible process. It can be concluded from the above table that a diet supplemented with a cysteine-rich peptide results in a quicker recovery to normal conditions. Example 9 · ACE inhibitory activity of a cysteine-rich peptide 99317.doc • 26 -200529866 Identification of ACE inhibition is based on Maguire and Price (Ann. Clin. 8T0 (: 1 ^ 〇1.1985, ¥ 〇1.22: 204-210) with furylpropionylphenylpropylamine-glycinyl- Glycinic acid (FAPGG) was used as the basis for matrix titration for micro-titration. ACE (nr. A6778), FAPGG (nr F7131), and Captopril® (ni · C8856) of rabbit lung were obtained from Sigma. Test substances of different concentrations ( Inhibitor) was added to the matrix solution (0.145 mmol), and the reaction was started by adding enzyme (ACE, 0.145 units). The decrease in absorbance was measured at 340 nm every 1 minute using the pQuant dish counter of Bio-Tek instruments. The curve of inhibitor concentration versus ACE inhibition (%) was used to determine the IC50. Whey protein isolate (Bipro, Davisco) was used as a negative control. IC50 was defined as the degree of inhibitory inhibition of ACE5%. Results: IC50 is rich in cysteine Glycine peptides:

203.23 mg / L 133.47 mg / L < 2 mg / L No inhibition 80 mg / L Reference CE 90 B is DMV -300% hydrolysate diafiltration (Example 1) -200% hydrolysate diafiltration (Example 1) Captopril®

Whey Protein Isolate (Bipro, Davisco) CE 90 B

Captopril® is a brewed protein hydrolysate produced by the well-known ACE inhibitor International (The Netherlands) and has ACE inhibitory activity. Example 1 (A study on the effects of cysteine-rich peptides made by humans. Thirteen people over 50 years old took cysteine peptide pills for 4 weeks at a dose of 99317.doc -27- 200529866. 3 · 3 g § cysteine-containing peptide mixture (equivalent to 200 mg cysteine / day). Before and after the test, the health status was studied by questionnaires and interviews. 9 people completed the study. 7 of them observed The positive effects are as follows: • Natural vitality has been increased • Better sleep quality (feeling fully rested after getting up early) • Improved mental alertness and better concentration • Two people with hypertension in this study reported lower blood pressure It is consistent with the result of Example 8. The effect of increasing vitality was written in writing 4-5 days after ingestion, and there was a report of decreased vitality after stopping taking pills. Exposure to stress at 10 different ages (30-50) ) People used the same dose of cysteine-rich peptides but did not complete the test. The observations reported by the testers were: • Sleep well (feeling adequate rest in the morning) and full of energy during the day. There are 5 Asian adults, I have said that I have a hangover Before drinking, Φ 0.8-1.6 g of peptide mixture rich in cysteine (containing cysteine 6.5% by weight) was taken. All individuals reported no hangover or experience To embarrassing flushing. ^ 2 Asian adults used 0.8-1.6 g of peptide mixture rich in cysteine (containing cysteine 6.5% by weight in the last 4 weeks) ). These individuals reported improvements in their skin condition and skin appearance (reduced acne). In addition, they reported more vitality and better sleep. 99317.doc -28-

Claims (1)

200529866 10. Scope of patent application: A peptide, β-cysteine, or peptide mixture containing at least 6.5% by weight of cysteine in the preparation of a drug, supplement, beverage or food that restores the internal balance of thiols The purpose of the product. 2. The use according to claim 1 for preventing and / or reducing the effects of drinking. The use of the cabinet according to claim 2 is to prevent and / or reduce hangovers. It is stated that the use of item 1 or 2 is for preventing and / or reducing facial flushing. 5. According to the purpose of claim 1, it is for improving vitality. 6. Use according to claim 6 for preventing and / or reducing fatigue. 7. The use according to claim 1, which is for improving sleep. 8. The use according to claim 1, which is for preventing and / or reducing metabolic symptoms, especially non-insulin-dependent diabetes mellitus, and the development of symptoms. 9. The use according to sunny demand item 8 for the prevention and / or reduction of the development of cardiovascular diseases (especially atherosclerosis). I. The use according to any one of claims 1 or 8 for reducing blood pressure. II. The use according to item 1 of the invention, which is for the prevention and / or treatment of poisoning caused by drugs. 12. The use according to claim 1, which is for whitening the skin. 13. Use according to claim 1, which is for reducing inflammation. 14. The use according to any one of claims 1 to 3, wherein the peptide containing the peptide mixture containing cysteine at 6.5% by weight is prepared by a method including the following steps: a) protein The source protein is cleaved into the peptide; b) the peptide obtained from the step 1) of the peptide digestion, the role of the peptide is 99317.doc 200529866 at least less than the peptide's cysteine site decays, thus forming a terminal Digested peptides of cysteine; C) Purified digested peptides. 15. A formulation for restoring thiol homeostasis in an individual in need, comprising an effective amount of a peptide mixture containing at least 6.5% by weight of cysteine. 16. The formulation according to claim 15 for preventing and / or reducing the effects of alcohol consumption on individuals in need thereof. 1 7. The formulation according to claim 16 for preventing and / or reducing hangovers. 1 8. The formulation according to claim 16 for preventing and / or reducing facial flushing. 1 9 · The formulation according to claim 15 for increasing vitality. 20. The formulation according to claim 19, for preventing and / or reducing fatigue. 21. The formulation according to claim 15 for improving sleep. 22. The formulation according to claim 15 for the prevention of the development of symptoms of metabolic symptoms (especially non-insulin-dependent diabetes mellitus). 23. The formulation according to claim 22, which is for the prevention and / or reduction of the development of cardiovascular diseases (especially atherosclerosis). 24. The formulation according to claim 15 for reducing blood pressure. 25. The formulation according to claim 15 for the prevention and / or treatment of drug-induced toxicity. 26. The formulation according to claim 15 for whitening the skin. 27. The formulation according to claim 15 for reducing inflammation. 28. The formulation according to any one of claims 15 to 25, wherein the peptide mixture, the peptide contains at least 6.5% by weight of cysteine, and is prepared by a method including 99317.doc 200529866 as follows Obtaining: a) the protein sequence of the protein source U) a ^ the coat is decomposed into peptides b) the protease is digested in half a half of the mashing step a) the effect of the protease minus the protease is to form a terminal Digested peptides that are less than half of the cysteine site of the peptide-cystine; c) Purified digested peptide. 99317.doc 200529866 VII. Designated representative map (1) The designated representative map in this case is: (none). (2) Brief description of the component symbols of this representative figure: 8. If there is a chemical formula in this case, please disclose the chemical formula that can best show the characteristics of the invention: (none) 99317.doc